The development of a thrombus is a highly complex process. In brief, the attachment of platelets to the vascular surface initiates the entire coagulation cascade, eventually leading to the aggregation of platelets covered with fibrin. The continuing fibrin deposition probably ceases within 24-48 hours from the beginning of the process, after which the thrombus will either be organized, i.e., eventually replaced with fibrous tissue composed of smooth muscle cells or fibroblasts, or fibrinolysis will take place. In the latter case, tissue plasminogen activator is secreted from the endothelium to bind to the fibrin on the thrombus where it catalyses the conversion of plasminogen to plasmin. The thrombus may then disintegrate as a result of fibrinolysis.
With the development of new thrombolytic agents (such as tissue plasminogen activator or streptokinase), rapid, reliable methods of diagnosing thrombosis are needed because there agents are most efficacious within 4-6 hours of thrombus formation (TIMI study group, N. Engl. J. Med. 312:932-936 (1985)). So far, biochemical parameters have not appeared to be of any help in solving this problem (G. E. Austin, Arch. Path. Lab. Med. 111:1158-1162 (1987)).
Scintigraphic methods of diagnosing thrombosis have been suggested. Initially, the reagents developed for the diagnosis were based on radiolabelled blood components (e.g., fibrinogen or platelets), for the reason that these components are incorporated into the thrombi under formation (for a review of these methods, see K. A. Krohn and L. C. Knight. "Radiopharmaceuticals for Thrombosis Detection: Selection, Preparation and Critical Evaluation", Seminars in Nuclear Medicine, Vol. VII, No. 3, July 1977, pp. 219-228). Some disadvantages of using labelled fibrinogen or platelets have been reported. In the case of radiolabelled platelets, A. M. Peters et al., British Medical Journal 293:1525 (Dec. 13, 1986), briefly point out that the production of labelled platelets is time-consuming and requires considerable technical skill for which reason its use has not become widespread. One of the most serious drawbacks is that the accumulation of the reagents in thrombi which are older than about 24 hours is presumably insufficient to permit scintigraphic detection (e.g., as briefly indicated by J. H. Paulsma-De Waal et al., NucCompact 18:284-286 (1987)).
More recently, attempts have been made to employ radiolabelled antibodies against a variety of blood components as reagents for the diagnosis of thrombosis. A considerable number of publications report the use of monoclonal or polyclonal antifibrin antibodies labelled with a variety of radioactive isotopes, .sup.111 In, .sup.131 I or .sup.99m Tc being the most commonly used isotopes, for the diagnosis of deep venous thrombosis in particular.
Thus, for instance, S. F. Rosebrough et at., Radiology 156:515-517 (1985), describe the use of .sup.131 I labelled monoclonal antifibrin antibodies for the imaging of venous thrombi induced in dogs. S. F. Rosebrough et al., Radiology 62:575-577 (1987), describe the use of .sup.131 I labelled monoclonal antibodies specific for human and dog fibrin for the imaging of mature thrombi, reporting the successful imaging of canine deep venous thrombi 1, 3 and 5 days old. Similarly, L. C. Knight et al., J. Nucl. Med. 29:494-502 (1988), describe the use of an .sup.111 In labelled monoclonal anti-fibrin antibody for imaging vascular thrombi in an animal (rabbit and dog) model, reporting the imaging of thrombi up to 4 days old in five out of eight rabbits, and of 0.5 hour- and 24 hour-old thrombi in six out of eight dogs. All these studies conclude that, based on these findings, labelled monoclonal antifibrin antibodies may also be useful for imaging thrombi in human patients.
Studies of the diagnosis of deep venous thrombosis in human beings by means of radiolabelled monoclonal antifibrin antibodies has been described by, for instance, M. Jung et al., Eur. J. Nucl. Med. 14(5-6):280-283 (1988); H. J. Aronen et al., Eur. J. Nucl. Med. 14(5-6):288-290 (1988); and F. De Geeter et al., Eur. J. Nucl. Med. 14(4-6):284-287 (1988). Jung et al. conclude that the scintigraphical analysis is useful for the diagnosis of established deep venous thrombosis of the calf, popliteal vein and thigh when the thrombi are not older than ten days (M. Jung et al., Eur. J. Nucl. Med. 14(5-6):280-283 (1988)). Aronen et al. report false positive results of the scintigraphic analysis and conclude that it is necessary to confirm a positive result by phlebography (H. J. Aronen et al., Eur. J. Nucl. Med. 14(5-6):288-290 (1988)). De Geeter et al., consider antifibrin scintigraphy to present a promising alternative to contrast venography (F. De Geeter et al., Eur. J. Nucl. Med. 14(5-6):284-287 (1988)).
Antifibrin antibodies have the advantage that their antigenic site is located in the thrombotic area. However, antifibrin antibodies have a long half-life in the circulation and consequently there is a high background activity. This limits the use of labelled antifibrin antibodies to non-emergency situations (cf. Z. Oster and P. Som, American Journal of Radiology 152:253-260 (1989), who also remark that labelled antifibrin antibodies may particularly well suited for the detection of mature deep venous thrombi).
Other publications disclose the use of radiolabelled monoclonal anti-platelet antibodies for the diagnosis of thrombosis. Thus, A. M. Peters et al., British Medical Journal 293:1525-1527 (1986), describe the use of an .sup.111 In labelled monoclonal anti-platelet antibody for imaging deep venous thrombi in patients and conclude that the antibody is useful for imaging fresh thrombi rather than mature thrombi because platelet adherence to the surface of the thrombus will have to be in progress in order to obtain a positive result of the imaging. They also suggest the use of the antibody for imaging renal allograft rejection, platelet uptake on prosthetic arterial surfaces and arterial and intracardiac thrombi.
A. W. J. Stuttle et al., Eur. J. Nucl. Med. 14(5-6):122-125 (1988), report the use of a fragment of a monoclonal antiplatelet antibody for imaging deep venous thrombi. Similarly, P. Som et al., J. Nucl. Med. 27:1315-1320 (1987), describe the use of .sup.99m Tc labelled monoclonal antiplatelet antibody fragments for imaging experimentally induced thrombi in dogs. They conclude that the antibody fragments can be used for imaging thrombi in the thorax without blood-pool subtraction, and that they may be useful for detecting intracoronary thrombi. They further indicate that the labelling of the antibody fragments is simpler than labelling platelets.
Z. H. Oster and P. Som. American Journal of Radiology 152:253-260 (February 1989), discuss the properties of labelled antiplatelet antibodies versus labelled antifibrin antibodies for the purpose of thrombus imaging. Relative to antifibrin antibodies which, as discussed above, have a long half-life, antiplatelet antibodies have the advantage of a rapid blood clearance. Their antigenic site is located on the circulating platelets, giving these the opportunity to be incorporated into the developing thrombus at an early stage, thereby increasing the target/blood ratio (i.e., the ratio between the activity at the thrombotic site and the background activity in the blood). On the other hand, the attachment of the antiplatelet antibodies to circulating platelets makes it difficult to visualize thrombi which are older than about 24 hours.
The utility of radiolabelled fibrinolytic enzymes (e.g., tissue plasminogen activator (t-PA), urokinase, streptokinase) or precursors therefor (e.g., plasminogen) for the detection of thrombi as well as for the localization of malignant tumors (in the case of t-PA) has also been studied for the reason of their affinity to fibrin or other components of thrombi. Thus, J. H. Paulsma-De Waal et al., op. cit., describe the study of Tc-labelled t-PA as a reagent for the detection of thrombosis. They conclude that systemically injected radiolabelled t-PA yields no useful results (i.e., activity uptake in the thrombus), presumably because of its rapid clearance in the liver, whereas locally injected labelled t-PA shows a distinct uptake of activity in the thrombus. On the other hand, D. J. Hnatowich et al., Eur. J. Nucl. Med. 13:467-473 (1987), have found positive imaging of experimentally induced canine thrombi with intravenously injected recombinant t-PA coupled to diethylenetriaminepentaacetic acid labelled with .sup.111 In, while still observing a rapid clearance of their reagent in the liver.
S. -L. Karonen et al., J. Nucl. Med. 29:1194-1199 (1988), report the localization of malignant tumors with radiolabelled recombinant t-PA, concluding that the accumulation of labelled t-PA in malignant tissue points to a potential use of the labelled t-PA for the detection of tumors. However, a later study (S. -L. Karonen et al., Eur. J. Nucl. Med. 14(5-6):610-611 (1988)) confirms that a major proportion of the radiolabelled t-PA is eliminated rapidly from plasma. Another disadvantage of using labelled t-PA is that because it accumulates in the liver, the risk of obtaining too high a local radioactive dosage makes it necessary to reduce the amount of diagnostic reagent employed. Apart from this, the labelled t-PA has to comprise with the t-PA formed by the body as a response to fibrin deposition for attachment sites on the thrombus.
Labelled streptokinase and urokinase have also been used for thrombus imaging, as reviewed by K. A. Krohn and L. C. Knight, op. cit., who also report some of the difficulties inherent in using either of these enzymes (vide p. 226) and conclude that they are not yet clinically useful for the localization of thrombi. The use of radiolabelled plasminogen has also been reviewed by K. A. Krohn and L. C. Knight, op. cit., who conclude that it is of potential use for imaging older thrombi.